Nicotinamide adenine dinucleotide (NAD) plays fundamental roles in both cellular energy metabolism and cellular signaling. In energy metabolism, the chemistry of the pyridine ring allows NAD to readily accept and donate electrons in hydride transfer reactions catalyzed by numerous dehydrogenases. Recent studies have revealed new roles for nicotinamide adenine dinucleotide (NAD) and its derivatives in transcriptional regulation (Lin and Guarente, Curr. Opin. Cell. Biol., (2003) 15, 241-246). The evolutionarily conserved Sir2 protein family requires NAD for its deacetylase activity and regulates a variety of biological processes, such as stress response, differentiation, metabolism, and aging. Despite its absolute requirement for NAD, the regulation of Sir2 function by NAD biosynthesis pathways is poorly understood in mammals.
NAD biosynthesis in vertebrates is markedly different from that of yeast and invertebrates (see FIG. 1). For example, vertebrates lack any obvious homolog of the yeast nicotinamidase (Pnc1) (Rongvaux et al., Bioessays (2003) 25, 683-690). Also, nicotinamide, rather than nicotinic acid, is the major substrate for NAD biosynthesis in mammals (Magni et al., Adv. Enzymol. Relat. Areas Mol. Biol. (1999) 73, 135-182). NAD biosynthesis from nicotinamide (and nicotinic acid) is conserved throughout vertebrates, including mammals. Furthermore, the recycling of nicotinamide into NAD is more direct in vertebrates (see FIG. 1B).
Nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nmnat) catalyzes two reactions in NAD biosynthesis. Nmnat catalyzes the conversion of NaMN to deamido-NAD, which in turn is converted to NAD by NAD synthetase. In mammals, Nmnat also plays a role in the recycling of NAD. NAD is reformed via the Nampt and Nmnat enzymes, which convert nicotinamide to nicotinamide mononucleotide (NMN) and then NMN to NAD, respectively (Emanuelli et al., J. Biol. Chem. (2001) 276, 406-412; Schweigler et al., FEBS Lett. (2001) 492, 95-100). It has been reported that nicotinamide administration to mammals causes an increase in NAD levels in tissues such as liver and kidney (Kaplan et al., J. Biol. Chem. (1956) 219, 287-298; Greengard et al., J. Biol. Chem. (1964) 239, 1887-1892).
Co-factors for NAD biosynthesis include PRPP, ATP, and MgCl2 (Magni et al., Adv. Enzymol. Relat. Areas Mol. Biol. (1999) 73, 135-182). PRPP is a substrate of Nampt and, along with nicotinamide, forms NMN. ATP is a co-substrate of Nmnat, donating adenine to synthesize NAD from NMN. In the Nmnat reaction, MgCl2 is necessary for optimal Nmnat activity (Emanuelli et al., J. Biol. Chem. (2001) 276, 406-412).
Both the Nampt and Nmnat enzymes have been identified and cloned, but the kinetic characteristics of the NAD biosynthesis pathway mediated by Nampt and Nmnat have not been determined. The Nmnat enzyme has been described in mouse (Conforti et al., Proc. Natl. Acad. Sci. USA (2000) 97, 11377-11382; Mack et al., Nat. Neurosci. (2001) 4, 1199-1206) and humans (Schweigler et al., FEBS Lett. (2001) 492, 95-100; Emanuelli et al., J. Biol. Chem. (2001) 276, 406-412; Fernando et al., Gene (2002) 284, 23-29). The bacterial Nampt enzyme was originally identified in Haemophilus ducreyi (Martin et al., J. Bacteriol. (2001) 183, 1168-1174) and found highly homologous to the human pre-B-cell colony-enhancing factor (PBEF), a presumptive cytokine whose cDNA was originally cloned in 1994 (Samal et al., Mol. Cell. Biol. (1994) 14, 1431-1437). Nampt enzymatic activity was detected from the mouse PBEF protein, whose cDNA was cloned in 2002 (Rongvaux et al., Eur. J. Immunol. (2002) 32, 3225-3234).
In mammals, the enzyme Sir2 acts upon NAD substrate to form nicotinamide and O-acetyl-ADP-ribose (Imai et al., Nature (2000) 403, 795-800; Moazed, Curr. Opin. Cell. Biol. (2001) 13, 232-238; Denu, Trends Biochem. Sci. (2003) 28, 41-48). Even though the [NAD]/[NADH] ratio modulates Sir2 function in skeletal muscle differentiation in mammals (Fulco et al., Mol. Cell. (2003) 12, 51-6213), it is uncertain whether NAD biosynthesis regulates Sir2 activity in these organisms. It has been suggested that nicotinamide plays a critical role as an endogenous inhibitor of Sir2 in yeast (Anderson et al., Nature (2003) 423, 181-185; Gallo et al., Mol. Cell. Biol. (2004) 24, 1301-1312).
There have been a number of studies to measure NAD biosynthesis. For example, NAD biosynthesis in erythrocytes has been measured (Micheli et al., Methods Enzymol. (1997) 280, 211-221). A liquid chromatographic-electrospray ionization ion trap mass spectrometry (LC/MS) method has been developed to measure the biosynthetic incorporation of specific precursors into NAD (Evans et al., Anal. Biochem. (2002) 306, 197-203). In addition, the Nmnat enzymatic activity has also been quantitatively measured (Balducci et al., Anal. Biochem. (1995) 228, 64-68; Emanuelli et al., J. Chromotogr. B. (1996) 676, 13-18; Emanuelli et al., J. Biol. Chem. (2001) 276, 406-412). Revollo et al. (2004) J. Biol. Chem. 279(49): 50754-50763 (published online on Sep. 20, 2004, doi:10.1074/jbc.M408388200)) is reported to have determined the enzymological parameters of Nampt and Nmnat.
NAD has been linked to age-associated diseases (Lin and Guarente, Curr. Opin. Cell. Biol., (2003) 15, 241-246) and carcinogenesis (Jacobson et al., Biochimie, (1995) 77, 394-398; Jacobson et al., Mol. Cell. Biochem. (1999) 193, 69-74). Regarding the connection to aging and age-associated diseases, Sir2 proteins have been demonstrated to play a role in regulating aging and longevity in lower eukaryotes, such as yeast, worms and flies (Blander and Guarente, Annu. Rev. Biochem. (2004) 73, 417-435; Wood et al., Nature (2004) 430, 686-689). Sir2 proteins are also required for the lifespan-extending effects of caloric restriction (Koubova and Guarente, Genes. Dev. (2003) 17, 313-321; Picard et al., Nature (2004) 429, 771-776; Cohen et al., Science (2004) 305, 390-392; Wood et al., Nature (2004) 430, 686-689). In mammals, Sir2 plays a role in mobilizing fat from adipose tissue (Picard et al., Nature (2004) 429, 771-776), protecting axons from injuries and toxic damages (Araki et al., Science (2004) 305, 1010-1013), and regulating insulin secretion in pancreatic β cells (Moynihan et al. Cell Metab. (2005) 2, 105-117).
Sir2 is also known to have anti-apoptotic effects (Luo et al., Cell (2001) 107, 137-148; Vaziri et al., Cell (2001) 107, 149-159; Motta et al., Cell (2004) 116, 551-563; Brunet et al., Science (2004) 303, 2011-2015) by enhancing cellular resistance to damages and stresses. NAD biosynthesis plays an important role in regulating Sir2 activity and thereby controls aging, at least in yeast (Anderson et al., J. Biol. Chem. (2002) 277, 18881-18890; Anderson et al., Nature (2003) 423, 181-185; Gallo et al., Mol. Cell. Biol. (2004) 24, 1301-1312)). The work reported herein also shows that NAD biosynthesis mediated by Nampt regulates Sir2 activity in mammals (see Revollo et al. (2004) J. Biol. Chem. 279(49): 50754-50763. Furthermore, it has recently been suggested that increasing NAD biosynthesis enhances Sir2 activity in neurons and may increase the resistance to neurodegenerative diseases (Araki et al., Science (2004) 305, 1010-1013).